Volume Electron Microscopy: What It Is and How It Works

Volume electron microscopy (vEM) is an imaging technology that generates detailed, three-dimensional models of biological samples with nanoscale resolution. It allows scientists to see the intricate structures within cells and tissues in their full 3D context. The process involves collecting a series of images from a sample and using computers to stack them into a complete model. This is comparable to creating a 3D model of a building by digitally stacking photographs of every floor, providing a comprehensive view that reveals how everything is connected.

Bridging the Gap from 2D to 3D Imaging

Traditional electron microscopes, such as transmission (TEM) or scanning (SEM), produce a single, flat, high-resolution image. These 2D images are detailed but only show a thin slice of a larger object, leaving out the context of surrounding structures. Life exists in three dimensions, and understanding it requires seeing more than a single plane. For example, a single image of a neural circuit cannot reveal how the neurons are intertwined and connected. Volume electron microscopy was developed to address this challenge, providing the tools to visualize the depth and complexity of biological systems.

Core Techniques of Volume Electron Microscopy

Volume electron microscopy methods can be grouped into two main approaches: those that slice the sample inside the microscope and those that slice it beforehand. Both approaches acquire a sequence of 2D images from successive layers of a sample, which computer software then aligns and reconstructs into a three-dimensional volume.

One category involves automated slicing inside the microscope’s vacuum chamber. In serial block-face scanning electron microscopy (SBF-SEM), an integrated diamond knife, or ultramicrotome, shaves a thin layer off a sample block. The microscope then scans the newly exposed face to create an image, and the process repeats automatically.

Another in-situ technique is focused ion beam scanning electron microscopy (FIB-SEM). Instead of a knife, FIB-SEM uses a focused beam of ions to mill a thin layer from the sample’s surface. After a layer is removed, the electron beam images the new surface. This process allows for thinner slicing than SBF-SEM, resulting in better resolution along the depth axis for smaller sample volumes.

The other approach involves sectioning the sample before it is placed in the microscope. With serial section transmission electron microscopy (ssTEM), a sample is cut into a ribbon of ultra-thin sections. These are collected on a support, imaged one-by-one in a transmission electron microscope, and the resulting digital images are computationally aligned to create the 3D reconstruction.

Preparing Samples for Viewing

Before a biological sample can be imaged, it must undergo a preparation process to preserve its structure and make it visible to the electron beam. This procedure transforms soft biological tissue into a stable state suitable for the conditions inside a microscope.

The first step is fixation, which uses chemicals to lock cellular components in place, preserving them in a near life-like state. Chemicals like glutaraldehyde and paraformaldehyde cross-link proteins and other molecules, preventing them from shifting or degrading.

Next, the sample is stained with solutions containing heavy metals, such as osmium, uranium, and lead. These electron-dense atoms block or scatter the microscope’s electron beam. Cellular structures bind these metals to varying degrees, creating the contrast needed to distinguish different parts of the cell.

Finally, the sample is embedded in a hard resin, such as epoxy. The fixed tissue is dehydrated and infused with the liquid resin, which is then hardened. This encases the tissue in a solid block, providing the mechanical support needed for it to be sliced into thin, uniform layers without distorting.

Applications in Scientific Discovery

The three-dimensional models from volume electron microscopy have led to discoveries across many fields of biology. By revealing the architecture of cells and tissues, vEM helps answer questions about how biological systems are built and function. This technology has been applied in neuroscience, cell biology, and developmental biology.

A primary application is in connectomics, which aims to map the complete wiring diagram of the brain. Using vEM, researchers can trace the paths of individual neurons through dense brain tissue and pinpoint the synapses that connect them. These reconstructions provide insights into how the brain processes information, forms memories, and learns, and helps in understanding both normal brain function and neurological disorders.

Volume electron microscopy also allows for the reconstruction of entire cells and their internal components, known as organelles. Scientists can create 3D models of structures like mitochondria or the endoplasmic reticulum. This provides a deeper understanding of how these organelles interact as a system and how their organization is disrupted by disease or viral infections.

In developmental biology, vEM provides 3D snapshots of how cells organize to build tissues and organs. Researchers can visualize the cellular arrangements and connections that emerge as an organism develops. This offers a clearer picture of the processes that guide the formation of a complete organism.